Picornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication involves one parental template whereas sexual RNA replication involves two or more parental templates. Sexual RNA replication mechanisms optimize the biological fitness of pathogens in enterovirus species A (EV-A71), species B (CVB3), species C (polio and CVA21) and species D (EV-D68). Our study will reveal how these viruses exchange genetic material during sexual RNA replication. Mankind could exploit these mechanisms to control or eradicate important pathogens, from polioviruses to rhinoviruses to EV-D68. Asexual template-dependent RNA replication, while efficient, renders viruses susceptible to error catastrophe, an overwhelming accumulation of mutations in viral RNA genomes incompatible with viability. Sexual RNA replication counteracts error catastrophe by purging mutations from viral RNA genomes. It remains uncertain how asexual and sexual RNA replication mechanisms work coordinately to maintain virus populations in nature. During the previous funding period, we discovered molecular features of the poliovirus polymerase required for sexual RNA replication mechanisms (aka viral RNA recombination). We were able to use these discoveries to specifically disable sexual replication mechanisms without impairing asexual replication mechanisms. Among our more striking findings is the relationship between sexual replication mechanisms and error catastrophe. When we disable sexual replication mechanisms, poliovirus becomes exquisitely sensitive to ribavirin-induced error catastrophe. These data substantiate long held theories regarding the advantages and disadvantages of asexual and sexual replication mechanisms among RNA viruses. Overarching hypothesis: Viral RNA recombination is a form of sexual replication that shapes & maintains picornavirus species groups and counteracts error catastrophe. During the next funding period, we plan to identify the features of viral polymerases required for sexual RNA replication. As outlined in this application, conserved features of the viral polymerase interact with nascent RNA products and RNA templates near the active site of the polymerase - providing a mechanism for viruses to distinguish between homologous and non-homologous partners in RNA recombination. The experiments outlined in this application will advance our understanding of picornavirus species groups, reveal molecular features of viral polymerases that maintain viral species groups in nature, further elucidate the antiviral mechanisms of ribavirin, and provide opportunities to control or eradicate important pathogens.